Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
Detection method:	Chemiluminescence
Detection range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Rat TNF- alpha in samples. No significant cross-reactivity or interference between Rat TNF- alpha and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat TNF- alpha. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat TNF- alpha and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat TNF- alpha, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat TNF- alpha. The concentration of Rat TNF- alpha in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	TNF-a: Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Homotrimer. Interacts with SPPL2B. Belongs to the tumor necrosis factor family.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Membrane protein, integral; Cytokine; Apoptosis Cellular Component: extracellular space; recycling endosome; cell surface; integral to plasma membrane; integral to membrane; plasma membrane; intracellular; external side of plasma membrane; phagocytic cup; lipid raft Molecular Function:identical protein binding; protease binding; cytokine activity; tumor necrosis factor receptor binding Biological Process: extracellular matrix organization and biogenesis; positive regulation of nitric oxide biosynthetic process; positive regulation of NFAT protein import into nucleus; activation of MAPK activity; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; positive regulation of caspase activity; positive regulation of translational initiation by iron; positive regulation of membrane protein ectodomain proteolysis; activation of NF-kappaB transcription factor; positive regulation of MAP kinase activity; cellular extravasation; tumor necrosis factor-mediated signaling pathway; positive regulation of phagocytosis; negative regulation of interleukin-6 production; JNK cascade; negative regulation of osteoblast differentiation; positive regulation of action potential; embryonic gut development; negative regulation of protein complex disassembly; response to drug; positive regulation of cytokine production; positive regulation of heterotypic cell-cell adhesion; positive regulation of mitosis; response to virus; positive regulation of interleukin-6 production; glucose metabolic process; negative regulation of cytokine secretion during immune response; positive regulation of protein transport; DNA damage response, signal transduction resulting in induction of apoptosis; response to mechanical stimulus; defense response to bacterium; positive regulation of transcription from RNA polymerase II promoter; skeletal muscle contraction; sequestering of triacylglycerol; positive regulation of JNK cascade; negative regulation of transcription from RNA polymerase II promoter; signal transduction; positive regulation of interleukin-18 production; chronic inflammatory response to antigenic stimulus; positive regulation of hair follicle development; negative regulation of cell proliferation; positive regulation of neuron apoptosis; protein kinase B signaling cascade; positive regulation of chronic inflammatory response to antigenic stimulus; inflammatory response; regulation of protein amino acid phosphorylation; transformed cell apoptosis; positive regulation of peptidyl-serine phosphorylation; humoral immune response; regulation of cell proliferation; positive regulation of protein kinase B signaling cascade; positive regulation of interferon-gamma production; positive regulation of programmed cell death; positive regulation of protein complex assembly; negative regulation of viral genome replication; response to hypoxia; regulation of insulin secretion; positive regulation of JNK activity; positive regulation of osteoclast differentiation; multicellular organismal development; response to glucocorticoid stimulus; response to lipopolysaccharide; positive regulation of NF-kappaB import into nucleus; osteoclast differentiation; regulation of immunoglobulin secretion; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of interleukin-8 biosynthetic process; positive regulation of chemokine production; cell activation; detection of mechanical stimulus involved in sensory perception of pain; organ morphogenesis; defense response to Gram-positive bacterium; induction of apoptosis via death domain receptors; positive regulation of transcription factor activity; negative regulation of L-glutamate transport; response to activity; negative regulation of transcription, DNA-dependent; leukocyte migration; apoptosis; positive regulation of smooth muscle cell proliferation; defense response; positive regulation of synaptic transmission; response to radiation; regulation of protein secretion; regulation of osteoclast differentiation; negative regulation of lipid catabolic process; lipopolysaccharide-mediated signaling pathway; regulation of I-kappaB kinase/NF-kappaB cascade; caspase activation; positive regulation of humoral immune response mediated by circulating immunoglobulin; positive regulation of protein complex disassembly; calcium-mediated signaling; MAPKKK cascade; negative regulation of glucose import; positive regulation of chemokine biosynthetic process; protein import into nucleus, translocation; positive regulation of protein kinase activity; positive regulation of fever; activation of MAPKKK activity; positive regulation of protein amino acid phosphorylation; receptor biosynthetic process; leukocyte tethering or rolling; negative regulation of myoblast differentiation; positive regulation of inflammatory response; positive regulation of cytokine secretion
NCBI Summary:	acts as a cytokine; binds TNF receptors; plays a role in regulation of cell proliferation, induction of apoptosis, and inflammatory response [RGD, Feb 2006]
UniProt Code:	P16599
NCBI GenInfo Identifier:	135938
NCBI Gene ID:	24835
NCBI Accession:	P16599. 1
UniProt Secondary Accession:	P16599,Q6EE11, Q9JI26, Q9JI27,
UniProt Related Accession:	P16599
Molecular Weight:	25,806 Da
NCBI Full Name:	Tumor necrosis factor
NCBI Synonym Full Names:	tumor necrosis factor
NCBI Official Symbol:	Tnf
NCBI Official Synonym Symbols:	Tnfa; RATTNF; TNF-alpha
NCBI Protein Information:	tumor necrosis factor; cachectin; tumor necrosis factor alpha; tumor necrosis factor (TNF superfamily, member 2); tumor necrosis factor ligand superfamily member 2
UniProt Protein Name:	Tumor necrosis factor
UniProt Synonym Protein Names:	Cachectin; TNF-alpha; Tumor necrosis factor ligand superfamily member 2; TNF-a
Protein Family:	Tumor necrosis factor
UniProt Gene Name:	Tnf
UniProt Entry Name:	TNFA_RAT

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	RLU	Average	Corrected
1000	53245 54105	53675	53650
500	22242 22736	22489	22464
250	10447 9975	10211	10186
125	4803 4999	4901	4876
62.5	2528 2378	2453	2428
31.25	1323 1239	1281	1256
15.63	681 735	708	683
0	25 25	25	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat TNF- alpha were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat TNF- alpha were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	53.29	136.93	449.48	49.99	147.84	445.02
Standard deviation	4.73	12.95	37.13	6.23	16.57	29.24
C V (%)	8.88	9.46	8.26	12.46	11.21	6.57

Recovery

The recovery of Rat TNF- alpha spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	86-101	92
EDTA plasma (n=5)	99-115	105
Cell culture media (n=5)	98-115	105

Linearity

Samples were spiked with high concentrations of Rat TNF- alpha and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	88-101	100-116	102-120
1:2	Average (%)	94	106	109
1:4	Range (%)	84-97	101-113	91-104
1:4	Average (%)	91	107	98
1:8	Range (%)	85-97	98-113	92-107
1:8	Average (%)	91	105	98
1:16	Range (%)	91-103	87-98	86-99
1:16	Average (%)	98	92	93

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.